Growth suppressive effect of pegylated arginase in malignant pleural mesothelioma xenografts.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a difficult-to-treat global disease. Pegylated arginase (BCT-100) has recently shown anti-tumor effects in hepatocellular carcinoma, acute myeloid leukemia and melanoma. This study aims to investigate the effects of PEG-BCT-100 in MPM. METHODS: A panel of 5 mesothelioma cell lines (H28, 211H, H226, H2052 and H2452) was used to study the in vitro effects of BCT-100 by crystal violet staining. The in vivo effects of BCT-100 were studied using 211H and H226 nude mice xenografts. Protein expression (argininosuccinate synthetase, ornithine transcarbamylase, cleaved PARP, cleaved caspase 3, cyclins (A2, D3, E1 and H), CDK4 and Ki67) and arginine concentration were evaluated by Western blot and ELISA respectively. Cellular localization of BCT-100 was detected by immunohistochemistry and immunoflorescence. TUNEL assay was used to identify cellular apoptotic events. RESULTS: Argininosuccinate synthetase was expressed in H28, H226, and H2452 cells as well as 211H and H266 xenografts. Ornithine transcarbamylase was undetectable in all cell lines and xenograft models. BCT-100 reduced in vitro cell viability (IC50 values at 13-24 mU/ml, 72 h) across different cell lines and suppressed tumor growth in both 211H and H226 xenograft models. BCT-100 (60 mg/kg) significantly suppressed tumor growth (p < 0.01) with prolonged median survival (p < 0.01) in both xenograft models. Combining BCT-100 with pemetrexed or cisplatin conferred no additional benefits over single agents. Serum and intratumoral arginine levels were effectively decreased by BCT-100, associated with cytosolic accumulation of BCT-100 within tumor cells. Apoptosis (PARP cleavage in 211H xenografts; Bcl-2 downregulation, and cleavage of PARP and caspase 3 in H226 xenografts; positive TUNEL staining in both) and G1 arrest (downregulation of cyclin A2, D3, E1 and CDK4 in 211H xenografts; suppression of cyclin A2, E1, H and CDK4 in H226 xenografts) were evident with BCT-100 treatment. Furthermore, proliferative factor Ki67 was downregulated in BCT-100 treatments arms. CONCLUSIONS: BCT-100 suppressed tumor growth with prolonged median survival partially mediated by intratumoral arginine depletion resulting in apoptosis and G1 arrest in mesothelioma xenograft models. The findings provide scientific evidence to support further clinical development of BCT-100 in treatment of MPM.

Inhibition of ornithine decarboxylase 1 facilitates pegylated arginase treatment in lung adenocarcinoma xenograft models.

Arginine depletion has shown anticancer effects among arginine auxotrophic cancers. An anti­proliferative effect of pegylated arginase (BCT­100) has been shown in acute myeloid leukaemia, hepatocellular carcinoma and mesothelioma. The aim of the present study was to evaluate the effect of BCT­100 in lung adenocarcinoma. A panel of lung adenocarcinoma cell lines and xenograft models were used to investigate the effect of BCT­100. Protein expression, arginine level, putrescine level, spermidine level and apoptosis were analyzed by western blotting, ELISA, high performance liquid chromatography, dot blot and TUNEL assay, respectively. BCT­100 converts arginine to ornithine. BCT­100 reduced in vitro cell viability across different lung adenocarcinoma cell lines and suppressed tumour growth in an HCC4006 xenograft, while paradoxical growth stimulation was observed in H358, HCC827, H1650 and H1975 xenografts. Upon BCT­100 treatment, ornithine decarboxylase 1 (ODC1) was induced in two solid tumour xenografts (H1650 and H1975). It was postulated that the accumulated ornithine could be channeled via ODC1 to produce polyamines that promoted tumour growth. The action of an ODC1 inhibitor (α­difluoromethylornithine, DFMO) was studied in the restoration of the anticancer effects of BCT­100 in lung adenocarcinoma. In both H1650 and H1975 xenografts, a combination of DFMO and BCT­100 significantly suppressed tumour growth, resulting in doubled median survival compared with the control. Putrescine was decreased in almost all treatment arms in the H1650, H1975 and HCC4006 xenografts. Nonetheless spermidine was reduced only following DFMO/BCT­100 treatment in the H1650 and H1975 xenografts. Apoptosis was enhanced in the combined treatment arm in both H1650 and H1975 xenografts. In the HCC4006 xenograft, addition of DFMO did not alter the tumour suppressive effect of BCT­100. In conclusion, inhibition of ODC1 by DFMO was crucial in facilitating BCT­100 treatment in lung adenocarcinoma that was partially mediated by depleting arginine and polyamines with consequent apoptosis.